RNA interference (RNAi) is a phenomenon in which mRNA is degraded by double-stranded RNA (dsRNA) and the like with specificity for the sequence thereof, resulting in suppression of gene expression. RNA interference has been shown to be conserved across various organisms, including nematodes, yeast and other fungi, insects, plants, and mammals, suggesting that it is a biological system common to all organisms.
Known biological roles of RNA interference include heterochromatin control in fission yeast and the like, control of DNA deletion in Tetrahymena and the like, and the like. It has been reported that deletion of Dicer (dcr1), Argonaute (ago1), or RdRp (rdp1) (these are genes playing important roles in the RNAi pathway) in fission yeast (mutants dcr1−, ago1−, and rdp1−, respectively) resulted in the aberrant accumulation of complementary transcripts from outer centromeric heterochromatic repeats, and this was accompanied by transcriptional de-repression of transgenes integrated at the centromere, loss of histone H3 lysine-9 methylation, and impairment of centromere function (Science, Vol. 297, pp. 1833-1837, 2002). Additionally, it was suggested that a short RNA derived from centromeric repeats is present in fission yeasts (Science, Vol. 297, p. 1831, 2002).
Because RNA interference enables the selective knock-down of a desired gene, it is highly expected to find new applications in biotechnological areas such as breed improvement of crop and medical areas such as gene therapy, as well as in basic sciences such as biochemistry.
There are two major methods of knocking down a gene by RNA interference: direct transfer of siRNA (short interfering RNA) into cells and transfer of siRNA expression vector into cells. Although the former method is quite simple, it is faulty in that the effect of the siRNA introduced does not persist for a long time when it is degraded. The latter siRNA expression vector method, on the other hand, is advantageous in that it enables the preparation of knockdown cell lines or knockdown animals thanks to the long persisting effect thereof. Because RNA interference in cells is triggered by the formation of double-stranded RNA, however, many siRNA expression vectors produce double-stranded RNAs such as hairpin RNAs, which in turn can cause the vector DNA itself to have a stem loop structure and hence become unstable in Escherichia coli; it has been difficult to construct a siRNA expression vector.